The effect of garlic extracts on the control of postharvest pathogens and postharvest decay of apples

Daniel, Chanel Karousha (2014-04)

Thesis (MSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Apples are an important export commodity for the South African market, and postharvest losses that occur as a result of decay due to infection with pathogenic fungi such as Botrytis cinerea Pers., Penicillium expansum (Link) Thom. and Neofabraea alba (E.J. Guthrie) are of major concern for all parties concerned with fruit production and distribution. Decay control of these fungi is primarily managed through the use of synthetic fungicides; however, pathogen development of resistance to these fungicides and recent worldwide concern over healthier living and a greener environment has called for the discriminate use of synthetic chemicals. This has opened up an avenue for the development of safer and more environmentally friendly alternatives to control postharvest decays. The use of plant extracts and essential oils are favoured as natural sources of antimicrobials whilst still being safe for human consumption and having no negative impact on the environment. Allium sativum (garlic) is one such plant species that is well documented for its value in improving human health and is readily available for consumption not just as a flavour component of food but also to be taken as a daily herbal diet supplement. Given the antimicrobial effectiveness of garlic against human pathogens and ailments, its value as an antifungal agent against postharvest pathogens causing grey mould, blue mould and bull’s eye rot of apples was investigated in vitro and in vivo within this study. Furthermore, an attempt was made to elucidate the chemical components of garlic extracts by gas chromatography-mass spectrometry (GC-MS). All experiments in this study were carried out with garlic extracts prepared from fresh garlic bulbs. For the in vitro experiments, two extract preparations of garlic, one containing ethanol (Extract 1) and one where ethanol had been removed by evaporation (Extract 2), was tested for antifungal action within an amended media experimental design. Both extract preparations were each subjected to two dilution series (0-80% garlic extract) with water and ethanol as diluents. Both extract preparations were successful at retarding pathogen mycelial growth and spore germination; however, concentrations of Extract 2 (ethanol evaporated) and diluted with distilled water provided markedly better inhibition of B. cinerea and P. expansum than the ethanolic dilutions of extract 2. Both extract preparations yielded similar inhibitory results when tested against N. alba. Due to the results achieved in the amended media experiments, the use of a crude garlic extract without ethanol and diluted in water was considered to be the best option for further tests throughout the remainder of the study. In vitro volatile effects of crude garlic extracts at concentrations between 0 and 40% garlic extract were subsequently tested. Garlic volatiles were effective in inhibiting pathogen mycelial growth and spore germination of all three pathogens, at lower concentrations compared to the amended media experiments. In vitro volatile exposure with garlic extracts was more effective at inhibiting N. alba than direct application of the extracts. Curative and protective application of garlic extracts and clove oil for increased fungal inhibition through synergism was tested by direct and volatile exposure to the pathogens in vivo on three economically important apple cultivars; ‘Granny Smith’, ‘Golden Delicious’, and ‘Pink Lady’. Direct exposure of artificially wounded and inoculated fruit to the garlic extract and clove oil revealed that garlic extracts applied curatively but not protectively effectively controlled decay caused by B. cinerea and P. expansum on all apple cultivars. Both curative and protective applications were ineffective in controlling N. alba. In vivo volatile exposure to the garlic extracts and clove oil did not inhibit decay on any of the cultivars and was not effective against any of the three pathogens investigated. A full chemical profile analysis was done by GC-MS analysis of garlic extract samples. The compounds diallyl disulphide, allyl methyl trisulphide, allyl methyl disulphide and dimethyl trisulphide were detected in relatively high amounts. This result suggests that the abundance of sulphur and sulphur related compounds detected may be responsible for the antifungal action noted in the experimental studies. In conclusion, garlic was shown to have antifungal activity against B. cinerea, P. expansum and N. alba. The pathogens used in this study were not compared with each other, but undoubtedly each pathogens reacts differently to exposure to the garlic extracts. It would therefore be advisable to investigate the effects of the extracts on each of the pathogens in a more in-depth study. More investigations into the application of the garlic extracts is required before it may be recommended for use; however, results for the use of garlic extracts against these postharvest pathogens and the postharvest decay they cause are promising.

AFRIKAANSE OPSOMMING: Appels is ‘n belangrike uitvoerproduk vir die Suid-Afrikaanse vrugtebedryf, maar noemenswaardige na-oes verliese word weens bederf deur patogeniese swamme soos Botrytis cinerea Pers., Penicillium expansum (Link) Thom. en Neofabraea alba (E.J. Guthrie) ervaar. Dit raak alle partye betrokke met die produksie en verspreiding van hierdie vrugsoort. Hierdie swamme word hoofsaaklik met behulp van kunsmatige swamdoders beheer, alhoewel weerstand-ontwikkeling en wêreldwye bewusmaking van ‘n gesonder leefstyl en omgewing die gebruik van kunsmatige middels streng aanspreek en die ontwikkeling van veiliger en meer omgewingsvriendelike alternatiewe middels verlang. Plant-ekstrakte en essensiële olies kan dien as sulke middels en is natuurlike bronne van anti-mikrobiese aktiwiteit, is veilig vir menslike verbruik en het ook geen negatiewe invloed op die omgewing nie. Allium sativum (knoffel) is so ‘n plantspesie wat as alternatiewe middel gebruik kan word. Dit is bekend vir sy waarde in die verbetering van menslike gesondheid, is maklik bekombaar en word nie net as ‘n geurmiddel vir voedsel gebruik nie, maar ook as ‘n daaglikse krui-aanvulling. Gegewe die anti-mikrobiese doeltreffendheid van knoffel teenoor menslike patogene en kwale, is die werking (in vitro en in vivo) teen na-oes patogene wat grys skimmel, blou skimmel en teikenvrot in appels veroorsaak, in hierdie studie ondersoek. Bepaling van die chemiese samestelling van die knoffel-ekstrak is ook met behulp van gaschromatografie massa spektrometrie (GK-MS) onderneem.Vars knoffelbolle is vir elke eksperiment in hierdie studie gebruik met die voorbereiding van die knoffel-ekstrak. Vir die in vitro eksperiment is twee knoffel-ekstrakte voorberei, naamlik: ‘n ekstrak wat etanol bevat (Ekstrak 1) en een waarvan die etanol verwyder is met verdamping (Ekstrak 2). Die ekstrakte is getoets vir werking teen fungi in kultuur-medium.. Albei ekstrakte is verdun tot twee konsentrasie reekse (0-80%) met water en etanol as verdunningsmiddels. Albei ekstrakte het suksesvolle werking getoon teenoor die patogene ten opsigte van vertraging van miseliumgroei en spoor-ontkieming, alhoewel konsentrasies van Ekstrak 2, verdun met gesuiwerede water, patogene B. cinerea en P. expansum beter onderdruk het as Ekstrak 2 verdunnings met etanol.. Beide ekstrakte en hul afsonderlike verdunnings met etanol en water het soortgelyke resultate gelewer met onderdrukking van N. alba. Volgens resultate wat verkry is van die kultuur-medium eksperimente, is Ekstrak 2 verdun met gesuiwerde water beskou as die geskikste vir verdere toetse in hierdie studie. Die vlugtige effek van Ekstrak 2 is in vitro getoets by konsentrasies tussen 0 tot 40%. Die vlugtige stowwe van knoffel het al drie patogene se groei en spoor-ontkieming effektief onderdrukby laer konsentrasies as wat gebruik is in die kultuur-medium eksperiment. Dus is in vitro blootstelling van N. alba aan die vlugtige stowwe meer effektief as direkte toediening van die ekstrakte. Die voorkomende en beskermende effek van die knoffel-ekstrak, asook naeltjie-olie, is in vivo ondersoek om te bepaal of die stowwe saam sterker onderdrukking van die patogene kon bewerkstellig. Direkte en vlugtige blootstelling is op drie ekonomies-belangrike appel-kultivars getoets, naamlik: ‘Granny Smith’, ‘Golden Delicious’ en ‘Pink Lady’. Direkte blootstelling met die knoffel-ekstrak en naeltjie-olie aan gewonde en ge-inokuleerde vrugte het aangedui dat B. cinerea- en P. Expansum-bederf net beheer kon word indien knoffel voorkomend toegedien is vir al die ondersoekte appel-variëteite. Voorkomende en beskermende toediening was onsuksesvolle om N. alba te beheer. In vivo blootstelling van die drie patogene aan die knoffel-ekstrak en naeltjie-olie se vlugtige stowwe kon nie enige van die patogene effektief onderdruk nie en was onsuksesvol in bederf-beheer. ‘n Volledige chemiese profiel is saamgestel deur GK-MS ontleding van die knoffelekstrakte. Hoë vlakke van verbindings dialliel disulfied, alliel-metiel-tri-sulfied, alliel-metieldisulfied en dimetiel-trisulfied is bespeur. Die aantal vrye sulfied en sulfied-verwante verbindings in die ekstrak kan moontlik ‘n verduideliking bied vir die anti-swam werking waargeneem gedurende hierdie studie. Ten slotte: knoffel toon ‘n anti-swam werking teenoor B. cinerea, P. expansum en N. alba. Die patogene in hierdie studie is nie met mekaar vergelyk nie, omdat elkeen uniek en uiteenlopend op knoffel reageer het. Alhoewel die huidige studie alreeds belowende resultate gelewer het, moet die ekstrak se effek op elke patogeen onderskeidelik nog in diepte ondersoek word, asook die wyse van die toediening in die na-oes praktyk voordat hierdie middel aanbeveel kan word vir gebruik.

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